Histidine ammonia lyase (EC 4.3.1.3) catalyzes the conversion of L-histidine to urocanic acid and ammonia. This is the first step in the degradation of histidine in both mammals and bacteria. A deficiency in this enzyme results in histidinemia, which is characterized by high serum histidine levels.
An isolated histidine ammonia lyase enzyme is one agent for treating increased histidine levels. Several lines of evidence indicate that in vivo depletion of serum histidine concentrations by histidine ammonia lyase could have additional therapeutic value. For example, histidine ammonia lyases have been shown in in vivo animal models to have potential therapeutic value against certain tumors. Roberts et al., Cancer Treat. Rep. 63:1045 (1979); Jack et al., Leukemia Res. 7:421 (1983).
Therapeutically useful (bioactive) enzymes generally display characteristics that are predictors of usefulness in vivo. These factors are outlined in Holcenberg and Roberts et al., Ann. Rev. Pharmacol. Toxicol. 17: 97 (1977), and include high activity at physiological pH and no requirement for exogenous cofactors. The histidine ammonia lyase isolated from a bacterium of the family Corynebacteriaceae, herein denoted as “HAL,” has been partially characterized by Roberts et al., Cancer Treat. Rep. 63: 1045 (1979). HAL demonstrates a broad useful pH range with approximately 75% of activity being retained at pH 7.2. The plasma half-life of HAL in mice is eight hours. The usefulness of this enzyme for histidine depletion in vivo is evident from the observation that single intraperitonial injections of 400 IU/kg effectively depleted plasma histidine in mice for up to 24 hours. However, the Corynebacteriaceae HAL which Roberts et al. described was not in purified form. As a result, many of the therapeutically beneficial properties associated with this HAL were unknown.
Histidine ammonia lyases have been isolated from several bacterial, animal, mammalian and plant sources. Shibatani et al., Eur. J. Biochem. 55: 263-269 (1975). Km values of these enzymes range between 1 and 20 mM. Shibatani (1975), supra; Wu et al., Gene. 115: 19-25 (1992); Jack et al., Leukemia Research, 7: 421-429 (1983); Khanna and Chang, Int'l J. Artificial Organs 13: 189-195 (1990). Genes coding for histidine ammonia lyases have been cloned from a number of organisms (Consevage, M. W. and A. T. Phillips. 1990. Journal of Bacteriology. 172 (5): 2224-2229; Oda, M. Sugishita, A. and K. Furukawa. 1988. J. Bacteriology. 170(7): 3199-3205; Wu, P. C., Kroening, T. A., White, P. J. and Kendrick, K. E. 1992. J. Bacteriology. 174(5): 1647-1655; Taylor, R. G., Lambert, M. A., Sexsmith, E., Sadler, S. J., Ray, P. N., Mahuran, D. J. and McInnes, R. R. 1990. J. Biol. Chem. 265(30): 18192-18199). Biochemical characterization has shown that most histidine ammonia lyases are inhibited by EDTA and thiol reagents (Shibatani, T., Kakimoto, T. and I. Chibata. 1975. Eur. J. Biochem. 55: 263-269; Okamura, H., Nishida, T. and H. Nakajawa. 1974. J. Biochem. 75: 139-152). A bioactive histidine ammonia lyase from a bacterium identified as Kurthia species was described by Jack, et al. in 1983 (Jack, G. W., Wiblin, C. N. and P. C. McMahon. 1983. Leukemia Research, 7(3): 421-429.) The Kurthia species histidine ammonia lyase was reported to have a Km of 1.25 mM with a half-life of 6-7 hours in mice. Chemical modification of the Kurthia histidine ammonia lyase did not increase the biological half-life of this enzyme. However, while HAL isolated from Corynebacteriaceae was effective in reducing ascites tumors in mice with high cell challenge (107 cells per mouse), the histidine ammonia lyase isolated from Kurthia was reported to be effective only at low tumor cell challenge levels (103 to 105 cells per mouse).
L-histidinol is an analog of histidine that is capable of altering histidine metabolism. Alteration of histidine metabolism by L-histidinol has provided therapeutic benefit. Histidine is required for several cellular processes, including protein synthesis and formation of histamine, both of which are required for tumor growth (Watanabe, et al, 1982. Biochem. and Biophys. Res. Comm. 109:478-485; Bartholeyns and Bouclier. 1982. Cancer Res. 44:639-645; Hakii, et al, 1986. J. Cancer Res. and Clin. Oncol. 111:177-181). Histidine is a direct precursor of histamine and is converted to histamine by the enzyme histidine decarboxylase (HDC). L-histidinol interferes with this conversion by inhibiting HDC. Therefore, L-histidinol can act therapeutically by inhibiting HDC, which is induced by strong tumor promoting phorbol esters (Mitra, et al, 1993. J. Cellular Physiol., 156:348-357). L-histidinol possesses some anti-tumor activity, as well as an ability to reverse resistance of certain tumor cell lines to some antineoplastic compounds (Stolfi, R. L. and Martin, D. S. 1990. Chemotherapy, 36 (6): 435-440; Warrington, R. C., Fang W. D. and L. U. Zhang, 1996. Anticancer Research 16 (6B):3641-3646; Warrington, R. C. and Fang W. D. 1989. Journal of the National Cancer Institute. 81 (10): 798-803). L-histidinol is also able to enhance the efficacy of certain anti-cancer drugs, when both are administered to a patient simultaneously. (Warrington, R. C. and W. D. Fang. 1991. Anticancer Research, 11 (5): 1869-1874; Warrington, R. C., Cheng, I. And W. D. Fang. 1994. Anticancer Research, 14 (2A): 367-372; Warrington, R. C., Cheng, I., Zhang, L. and W. D. Fang. 1993. Anticancer Research, 13 (6A): 2107-2112; Warrington, R. C. 1992. Biochemistry and Cell Biology, 70 (5): 365-375; Zaharko, D., Plowman, J., Waud, W., Dykes, D. and L. Malspeis. 1992. Cancer Research, 52 (13): 3604-3609). For example, the therapeutic index of chemotherapeutic agents is increased by combining treatment with L-histidinol, since L-histidinol reduces the toxicity of normal chemotherapeutic agents to normal cells but not to cancer cells (Warrington, R. C., Fang, W. D., Zhang, L. Shieh, M. and M. H. Saier, Jr. 1996. Anticancer Research, 16 (6B): 3635-3639; Warrington, R. C., Fang W. D., Zhang, L., Shieh, M. and M. H. Saier, Jr. 1996. Anticancer Research, 16 (6B): 3629-3633; Badary, O. A., Nagi, M. N., Al-Sawaf, H. A, Al-Harbi, M., and A. M. Al-Bekairia. 1997. Nephron, 77 (4): 435-439; Al-Shabanah, O. A., Badary, O. A., Al-Gharably, N. M. and H. A. Al-Sawaf. 1998. Pharmacological Research, 38 (3): 225-230; Badary, O. A. 1999. Experimental Nephrology, 7 (4): 323-327).
In theory, the use of L-histidinol with a histidine ammonia lyase offers a therapeutic approach to depleting serum histamine and lowering histidine levels. L-histidinol has limited usefulness as a single agent due to its low half-life (Zaharko, D., Plowman, J., Ward, W., Dykes, D., and L. Malspeis, 1992. Cancer Research. 52: 3604-3609) and its mode of action as a competitive inhibitor. Accordingly, L-histidinol must be present in very high concentrations in order to competitively inhibit cellular processes involving histidine. Reduced histidine levels would enhance the effectiveness of L-histidinol, by allowing cells to uptake the L-histidinol more readily.
Nevertheless, a histidine ammonia lyase suitable for combination therapy with a histidine analog, such as L-histidinol, must have the additional property of not being inhibited by L-histidinol. One prevalent characteristic of all known isolated histidine ammonia lyases is their inhibition in the presence of a histidine analog, like histidinol. For example, histidine ammonia lyases isolated from bacteria such as Achromobacter liquidum and Streptomyces griseus are inhibited by L-histidinol and L-histidinol phosphate, respectively, with a Ki of 4.58 and 0.27 mM, respectively (Shibatani, T. et al. 1975. Eur. J. Biochem. 55: 263-269; Wu, P. C. et al. 1995. Gene. 115(1-2): 19-25).
Due to their enzymatic inhibition by histidinol, previously described histidine ammonia lyases have not been suitable candidates for use in combination therapies with these histidine analogs for treating pathologies such as cancer. Accordingly, there is a present and unmet need for a histidine ammonia lyase that possesses the relevant properties associated with previously isolated histidine ammonia lyases, yet maintains the ability to deplete histidine in the presence of L-histidinol.
In addition to cancer, viral diseases such as Human Respiratory Syncytial Virus (RSV), Herpes Simplex Virus (HSV) and Human Immunodeficiency Virus (HIV), infect millions worldwide and cause major health problems. RSV, a common cause of winter outbreaks of acute respiratory disease, in 1998 resulted in 90,000 hospitalizations and 4,500 deaths and is the largest cause of lower respiratory tract disease among infants and young children in the United States (CDC. 1997. MMWR. 46(49); 1163-1165). Herpes Simplex Virus infects an even larger portion of the population. The Centers for Disease Control estimated that in 1998, 45 million people ages 12 and older, or one out of five of total adolescent and adult population, was infected with the Herpes Simplex Virus. The Joint United Nation Programme on HIV/AIDS (UNAIDS) estimates that worldwide 33.6 million persons are infected with HIV/AIDS and 2.6 million people died in 1999 from this disease.
Human infectious viruses vary widely in the way they enter cells, replicate inside the cells, and subsequently get released from infected cells. RNA viruses have single- or double-stranded RNA as their genomes, which are naked or enveloped. The RNA strand can be either in a positive or negative form. RNA viruses enter the cell, make copies of their RNA genome, and direct the synthesis of messenger RNA to code for structural and regulatory proteins. Finally, the genome is assembled with structural proteins and the virus is released. DNA viruses have single- or double-stranded DNA genomes that can be either non-enveloped or enveloped. Retroviruses are also RNA viruses but they involve DNA in their replication process. Thus, each virus is unique in its infection and multiplication process.
One common theme in viral replication is the ability of a virus to utilize the human cellular machinery for its multiplication. This makes drug development against viruses very difficult. In the past, antiviral therapy has focused on development of appropriate vaccines or inhibiting unique processes in viral replication. This often renders such therapy very specific for a type or subtype of viruses. Currently, vaccines are the main line of defense against viruses. Vaccines are developed specifically for each virus type and subtype, and are useful only against that particular virus type/subtype.
Therapies also have been developed that take advantage of unique processes in viral replication. For example, reverse transcriptase is unique to retroviruses. Nucleotide analogs and non-nucleotide reverse transcriptase inhibitors have been developed that inhibit reverse transcriptase without affecting other polymerases. However, such therapy is limited to combating only retroviruses. Yet another approach that targets a unique viral replication process is the use of protease inhibitors against HIV. But since these inhibitors target a specific enzyme, HIV protease, they cannot be effective against a wide range of viruses. Yet another example of a virus-specific therapy is the use of the antiviral compound ganciclovir, which is effective against Herpes Simplex Virus. Ganciclovir is specifically cytotoxic to herpes infected cells. Although ganciclovir therapy may be beneficial to combating the Herpes Simplex Virus, it has limited or no application for treating other viruses.
Accordingly, there is a great need for a therapeutic agent that can be effective against a broad spectrum of viruses. There has been no indication heretofore that a peptide having a histidine ammonia lyase activity could effectively treat infectious viral agents. Thus, a substantial therapeutic and market potential exists for a histidine ammonia lyase that is effective against infectious viral agents.